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transfected into eukaryotic cells. Any expression of the reporter gene will be driven by

the foreign DNA which must therefore contain promoter sequences (Fig. 6.42). These

plasmids and other reporter genes such as those usinggreen fluorescent protein(GFP)

or the firefly luciferase gene allow quantitation of gene transcription in response to

transcriptional activators.

The binding of a regulatory protein or transcription factor to a specific DNA site results

in a complex that may be analysed by the technique termedgel retardation. Under gel

electrophoresis the migration of a DNA fragment bound to a protein of a relatively large

mass will be retarded in comparison to the DNA fragment alone. For gel retardation to be

useful the region containing the promoter DNA element must be digested or mapped with

a restriction endonuclease before it is complexed with the protein. The location of the

promoter may then be defined by finding the position on the restriction map of the

fragment that binds to the regulatory protein and therefore retards it during electrophor-

esis. One potential problem with gel retardation is the ability to define the precise

nucleotide binding region of the protein, since this depends on the accuracy and detail

of the restriction map and the convenience of the restriction sites. However it is a useful

first step in determining the interaction of a regulatory protein with a DNA binding site.

DNA footprinting relies on the fact that the interaction of a DNA-binding

protein with a regulatory DNA sequence will protect that DNA sequence from degrad-

ation by an enzyme such as DNase I. The DNA regulatory sequence is first labelled

at one end with a radioactive label and then mixed with the DNA-binding protein

Promoter CAT gene

pCAT

Transcription and translation

pCAT

CAT protein

Transfection

of cells

Incubate CAT protein 37°C

[^14 C]chloramphenicol acetyl-CoA Lyse cells

Inactive promoter Active promoter

Aceylated

chloramphenicol

Chloramphenicol

Autoradiograph

Fig. 6.42Assay for promoters using the reporter gene for chloramphenicol acetyl transferase (CAT).

245 6.8 Analysing genes and gene expression